Affiliation: Kristian Gerhard Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACTMineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

pone.0120374.g002: SEM micrographs of the alginate network in ALP-modified (A, C, E) or unmodified (B, D, F) beads taken at day 21 post encapsulation.Cells were cultured in either growth medium (A, B) or osteogenic medium (C-F). Mineral particles with spherical morphology are clearly visible for heavily mineralized samples shown at high and low magnification in panel C and E.

Mentions:
Mineralization of alginate beads can be observed by light microscopy as the beads become more opaque and appear dark under transmitted light [17]. Hence, we examined the beads with encapsulated MSC by light microscopy to evaluate mineralization at day 2 and 21 post encapsulation (Fig. 1). A total of 4 samples were studied: ALP-modified and unmodified beads cultured in either growth medium or osteogenic medium. At day 2 post encapsulation unmodified beads appeared relatively transparent (Fig. 1A). ALP-modified beads were mineralized as they appeared darker (Fig. 1B). Interestingly, beads which were initially ALP-modified were then further mineralized during culture in both osteogenic medium and growth medium (Fig. 1B). The unmodified beads, however, did only mineralize when they were cultured further in osteogenic medium, but to a lower extent than the ALP-modified beads. These findings suggest that MSC differentiate into osteogenic cells capable of inducing matrix mineralization in both modified and unmodified alginate beads. To examine the material in the beads in more detail, we examined beads that had been cultured for 21 days in vitro using scanning electron microscopy (SEM) (Fig. 2) We have previously shown that mineralization of alginate hydrogels can be characterized with SEM [17, 19]. In general, we observe mineral crystals in the samples that appeared mineralized by light microscopy. Hence, ALP-modified beads cultured in osteogenic medium had highest level of mineralization, showing distinctive granular crystal morphology (Fig. 2C and B). This is followed by the ALP-modified sample cultured in growth medium (Fig. 2A) and the unmodified beads cultured in osteogenic medium, where only some evidence of mineralization could be detected (Fig. 2D and F). In the unmodified sample cultured in growth medium no crystals could be seen either in LM or SEM. (Fig. 1A and 2B).

pone.0120374.g002: SEM micrographs of the alginate network in ALP-modified (A, C, E) or unmodified (B, D, F) beads taken at day 21 post encapsulation.Cells were cultured in either growth medium (A, B) or osteogenic medium (C-F). Mineral particles with spherical morphology are clearly visible for heavily mineralized samples shown at high and low magnification in panel C and E.

Mentions:
Mineralization of alginate beads can be observed by light microscopy as the beads become more opaque and appear dark under transmitted light [17]. Hence, we examined the beads with encapsulated MSC by light microscopy to evaluate mineralization at day 2 and 21 post encapsulation (Fig. 1). A total of 4 samples were studied: ALP-modified and unmodified beads cultured in either growth medium or osteogenic medium. At day 2 post encapsulation unmodified beads appeared relatively transparent (Fig. 1A). ALP-modified beads were mineralized as they appeared darker (Fig. 1B). Interestingly, beads which were initially ALP-modified were then further mineralized during culture in both osteogenic medium and growth medium (Fig. 1B). The unmodified beads, however, did only mineralize when they were cultured further in osteogenic medium, but to a lower extent than the ALP-modified beads. These findings suggest that MSC differentiate into osteogenic cells capable of inducing matrix mineralization in both modified and unmodified alginate beads. To examine the material in the beads in more detail, we examined beads that had been cultured for 21 days in vitro using scanning electron microscopy (SEM) (Fig. 2) We have previously shown that mineralization of alginate hydrogels can be characterized with SEM [17, 19]. In general, we observe mineral crystals in the samples that appeared mineralized by light microscopy. Hence, ALP-modified beads cultured in osteogenic medium had highest level of mineralization, showing distinctive granular crystal morphology (Fig. 2C and B). This is followed by the ALP-modified sample cultured in growth medium (Fig. 2A) and the unmodified beads cultured in osteogenic medium, where only some evidence of mineralization could be detected (Fig. 2D and F). In the unmodified sample cultured in growth medium no crystals could be seen either in LM or SEM. (Fig. 1A and 2B).

Bottom Line:
Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes.In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture.Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.

Affiliation:
Kristian Gerhard Jebsen Center for Myeloma Research, Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

ABSTRACTMineralized biomaterials are promising for use in bone tissue engineering. Culturing osteogenic cells in such materials will potentially generate biological bone grafts that may even further augment bone healing. Here, we studied osteogenic differentiation of human mesenchymal stem cells (MSC) in an alginate hydrogel system where the cells were co-immobilized with alkaline phosphatase (ALP) for gradual mineralization of the microenvironment. MSC were embedded in unmodified alginate beads and alginate beads mineralized with ALP to generate a polymer/hydroxyapatite scaffold mimicking the composition of bone. The initial scaffold mineralization induced further mineralization of the beads with nanosized particles, and scanning electron micrographs demonstrated presence of collagen in the mineralized and unmineralized alginate beads cultured in osteogenic medium. Cells in both types of beads sustained high viability and metabolic activity for the duration of the study (21 days) as evaluated by live/dead staining and alamar blue assay. MSC in beads induced to differentiate in osteogenic direction expressed higher mRNA levels of osteoblast-specific genes (RUNX2, COL1AI, SP7, BGLAP) than MSC in traditional cell cultures. Furthermore, cells differentiated in beads expressed both sclerostin (SOST) and dental matrix protein-1 (DMP1), markers for late osteoblasts/osteocytes. In conclusion, Both ALP-modified and unmodified alginate beads provide an environment that enhance osteogenic differentiation compared with traditional 2D culture. Also, the ALP-modified alginate beads showed profound mineralization and thus have the potential to serve as a bone substitute in tissue engineering.